Ab120487

All enzymatic assays were carried out in HEPES buffer (50 mM Hepes, 50 mM NaCl, 0.1% CHAPS, 10 mM EDTA, 5% glycerol, 10 mM DTT, pH = 7.2) supplemented with 0.02% Triton X-100 at 25 °C for recombinant caspases, and at 37 °C for lysates. Appropriate enzyme/lysate dilutions were added as indicated, to reaction solutions containing our probes at a final concentration of 6 μM in 384-well microplate (Greiner Bio-One #781900). Control experiments with commercial tetrapeptide-AMC substrates were done where indicated. For inhibitor treatments, Z-VAD(OMe)-FMK (50 μM, ab120487, Abcam) or caspase-3/7 inhibitor I (200 μM for tissue lysate, Calbiochem #218826) was pre-incubated with biological samples for 3 h at 4 °C, prior to incubation with the probe. Liberation of fluorescence (λ_ex = 360 ± 40 nm; λ_em = 528 ± 20 nm for AAN, and λ_em = 460 ± 40 nm for AMC and DAN) was recorded by using a BioTek Synergy 4 plate reader. Kinetic constants were computed by fitting the data to Michaelis-Menton equation using a non-linear regression via software Origin™. The error bar in graphs or “±” in the table is a standard deviation (s.d.) of the data.

Hydroxyurea (3 mM for 2 h for QIBC and immunoblotting, 0.5 mM, 1 mM, or 2 mM for 24 h for clonogenic assays, H8627; Sigma-Aldrich), UCN-01 (300 nM for 1 h, U6508; Sigma-Aldrich), APH (50 μg/ml for 2 h, A4487; Sigma-Aldrich), thymidine (2.2 mM for 12 or 17 h, T1895; Sigma-Aldrich), nocodazole (100 ng/ml for 12 h, M1404; Sigma-Aldrich), PD0332991 (5 μM for 12 h, PZ0383; Sigma-Aldrich), TAK-931 (300 nM for 12 h, HY-10088; Biotech), bleomycin (25 μg/μl for 3 h on and 3 h off, S1214; Stratech), CPT (500 nM for 6 h, C9911; Sigma-Aldrich), Z-VAD-FMK (50 μM, ab120487; Abcam). Protein expression was induced in with 0.5–1 μg/ml tetracycline for 24 h (T7660; Sigma-Aldrich).

Human U2OS and HCT116 cells were obtained from ATCC. All cell lines used in this study were cultured in DMEM containing 10% FBS and were regularly tested negative for mycoplasma infection. To generate U2OS or HCT116 cell lines inducibly expressing GFP‐tagged or untagged FAM111A as well as GFP‐FAM111B WT and mutant alleles, cells were co‐transfected with pcDNA4/TO/GFP or pcDNA4/TO expression constructs and pcDNA6/TR (Invitrogen). Positive clones were selected by incubation in medium containing 5 μg/ml blasticidin S (Invitrogen) and 400 μg/ml Zeocin (Invitrogen) for 14 days. To generate cell lines with targeted knockout of FAM111A and/or FAM111B (ΔFAM111A, ΔFAM111B, and ΔFAM111A + ΔFAM111B), parental U2OS cells were transfected with pX459‐sgFAM111A#2 (ΔFAM111A), pX459‐sgFAM111B#2 (ΔFAM111B), or a 1:1 mix of pX459‐sgFAM111A#2 and pX459‐sgFAM111B#2 (ΔFAM111A+ΔFAM111B) and selected briefly with puromycin during clonal selection. Clones were screened for FAM111A and FAM111B expression by immunoblotting. Unless otherwise indicated, the following drug concentrations were used: nocodazole (10 μM, Sigma‐Aldrich), Z‐VAD‐FMK (50 μM, ab120487, Abcam), doxycycline (1 μg/ml, Sigma‐Aldrich), and AEBSF (5 mM, Sigma‐Aldrich).